ABSTRACT The overall goal of this Phase II project is to validate novel leishmanial protein biomarkers for development of a non-invasive urine-based assay to diagnose active visceral leishmaniasis (VL) and to monitor the therapeutic efficacy of this serious disease. VL is endemic in 47 countries, affects 500,000 people a year and kills more than 50,000, 70% of them children under age 15. VL, also known as kala-azar, is caused by parasites of the Leishmania donovani complex: L. donovani and L. archibaldi in the Old World (primarily India and South Eastern Africa), and L. infantum in the New World (Southern Europe and South America). Global VL morbidity and mortality in many parts of the world are increasing due to co-infection with human immunodeficiency virus. Although VL is usually fatal if not treated promptly, the effective drugs are toxic, expensive and difficult to administer, and untreated people with VL are reservoirs of infection who put others in their communities at risk. The gold standard for diagnosis is observation of the parasites (or detection of parasite DNA) in spleen, liver, lymph node or bone marrow aspirates; serum tests measure anti- parasite antibodies, which cannot distinguish between active VL from either prior exposure to the parasite or subsequent to successful treatment of the disease. There is no vaccine for human VL. The WHO has defined that a key requirement for effective control of this serious worldwide disease is a sensitive non-invasive test that can rapidly and reliably diagnose active VL and identify people who need immediate treatment. Our former published work and the Phase I component of this Phase II application have established the foundation for the development of a simple non-invasive urine test to both diagnose active VL and to monitor the therapy efficacy of this disease. We used mass spectroscopy to initially identify three L. infantum and more recently four new L. donovani proteins excreted in the urine of VL patients. We characterized these antigens, raised polyclonal antibodies against them and developed an antigen detection capture ELISA to diagnose VL. A pilot clinical study defined that the three proteins of L. infantum were present in the urines 19/20 well characterized New World VL patients and in none of more than 60 control urines samples from healthy subjects as well as from non VL patients like suffering from cutaneous leishmaniasis, Chagas? disease, schistosomiasis and tuberculosis. We are currently validating the recently discovered L. donovani markers. For this Phase II proposal we will use a large panel of urine samples from different areas of the world where VL is endemic to validate all seven discovered L. infantum and L. donovani biomarkers as reliable tools for the accurate diagnosis of active VL and to monitor the therapy of this disease. In addition to a solid preliminary data, a strength of this proposal is our access to a unique resource of urine samples from confirmed VL patients from VL endemic regions around the world.